Antifungal susceptibility of Candida biofilms: unique efficacy of amphotericin B lipid formulations and echinocandins

Abstract

Biofilms, likely the predominant mode of device-related microbial infection, exhibit resistance to antimicrobial agents. Evidence suggests that Candida biofilms have dramatically reduced susceptibility to antifungal drugs. We examined antifungal susceptibilities of Candida albicans and Candida parapsilosis biofilms grown on a bioprosthetic model. In addition to conventional agents, we determined if new antifungal agents (triazoles, amphotericin B lipid formulations, and echinocandins) have activities against Candida biofilms. We also explored effects of preincubation of C. albicans cells with subinhibitory concentrations (sub-MICs) of drugs to see if they could modify subsequent biofilm formation. Finally, we used confocal scanning laser microscopy (CSLM) to image planktonic- and biofilm-exposed blastospores to examine drug effects on cell structure. Candida biofilms were formed on silicone elastomer and quantified by tetrazolium and dry weight (DW) assays. Susceptibility testing of fluconazole, nystatin, chlorhexidine, terbenafine, amphotericin B (AMB), and the triazoles voriconazole (VRC) and ravuconazole revealed resistance in all Candida isolates examined when grown as biofilms, compared to planktonic forms. In contrast, lipid formulations of AMB (liposomal AMB and AMB lipid complex [ABLC]) and echinocandins (caspofungin [Casp] and micafungin) showed activity against Candida biofilms. Preincubation of C. albicans cells with sub-MIC levels of antifungals decreased the ability of cells to subsequently form biofilm (measured by DW; P < 0.0005). CSLM analysis of planktonic and biofilm-associated blastospores showed treatment with VRC, Casp, and ABLC resulted in morphological alterations, which differed with each agent. In conclusion, our data show that Candida biofilms show unique susceptibilities to echinocandins and AMB lipid formulations.

FIG. 1.

Activities of different concentrations of various antifungal agents against C. albicans biofilms. Graph shows XTT activity of C. albicans strain M61 under treatment with various concentrations of antifungal agents including FLC, AMB, Casp, and ABLC. Results were normalized to control (untreated) C. albicans strain M61, which was taken as 100%. Each result is representative of at least two experiments. For details of methods used, see text.

FIG. 2.

Effects on biofilm production of preincubation of C. albicans with subinhibitory concentrations of antifungal agents. Graph shows XTT (white bars) and DW (black bars) assay results with C. albicans strain M61 treated with one-fourth MIC₅₀s of various antifungal agents. Results were normalized to control (untreated) C. albicans strain M61, which was taken as 1 (100%). Assays were performed in quadruplicate. Each result is representative of at least two experiments. All values are means plus standard deviations. Comparisons are significant for P values of <0.0083. ∗, P < 0.0005 in DW assay, compared to untreated M61.

FIG. 3.

CSLM of planktonic C. albicans cells treated with antifungal agents. Images utilize CAAF and FUN-1 staining, a 63× oil immersion objective, and 2× magnification. Green CAAF staining highlights blastospore cell walls. FUN-1 is yellow in metabolically inactive, nonviable cells; viable cells convert the stain into red-fluorescing aggregates. (A to D) Effects on planktonic C. albicans of treatment with Casp (B), ABLC (C), and VRC (D) compared with untreated control cells (A). Cells were exposed to antifungal agents for 24 h. Cell clumping is an artifact of the cytospin preparation. Arrows point to markedly distorted cells

FIG. 4.

CSLM of biofilm-associated C. albicans cells treated with antifungal agents. Images utilize CAAF and FUN-1 staining, a 63× oil immersion objective. Magnification, ×30. Each image is a Z-slice (in the z axis) of the basal blastospore layer. Green CAAF staining highlights blastospore cell walls. FUN-1 is yellow in metabolically inactive, nonviable cells; viable cells convert the stain into red-fluorescing aggregates. Shown are the effects on biofilm-associated C. albicans (specifically, the basilar blastospore layer) of Casp (B), ABLC (C), or VRC (D), compared with untreated control cells (A). Biofilm was grown for 48 h and then exposed to antifungals for an additional 48 h. Arrows point to red FUN-1 aggregates, indicating viable cells.